Diversity of zooplankton assemblages at Station L4, Western English Channel, measured using next generation DNA sequencing

Background: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel Next Generation Sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify the richness and diversity of a mixed zooplankton assemblage from a productive monitoring site in the Western English Channel. Methodology/Principle Findings: Plankton WP2 replicate net hauls (200 µm) were taken at the Western Channel Observatory long-term monitoring station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,042 sequences were obtained for all samples. The sequences clustered in to 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 138 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 75 taxonomic groups. Conclusions: The percentage of OTUs assigned to major eukaryotic taxonomic groups broadly aligns between the metagenetic and morphological analysis and are dominated by Copepoda. However, the metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for estimating diversity and species richness of zooplankton communities.

Molecular composition in surface and subsurface sediments of the Helgoland mud area, North Sea

The role of microorganisms in the cycling of sedimentary organic carbon is a crucial one. To better understand relationships between molecular composition of a potentially bioavailable fraction of organic matter and microbial populations, bacterial and archaeal communities were characterized using pyrosequencing-based 16S rRNA gene analysis in surface (top 30 cm) and subsurface/deeper sediments (30-530 cm) of the Helgoland mud area, North Sea. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) was used to characterize a potentially bioavailable organic matter fraction (hot-water extractable organic matter, WE-OM). Algal polymer-associated microbial populations such as members of the Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia were dominant in surface sediments while members of the Chloroflexi (Dehalococcoidales and candidate order GIF9) and Miscellaneous Crenarchaeota Groups (MCG), both of which are linked to degradation of more recalcitrant, aromatic compounds and detrital proteins, were dominant in subsurface sediments. Microbial populations dominant in subsurface sediments (Chloroflexi, members of MCG, and Thermoplasmata) showed strong correlations to total organic carbon (TOC) content. Changes of WE-OM with sediment depth reveal molecular transformations from oxygen-rich [high oxygen to carbon (O/C), low hydrogen to carbon (H/C) ratios] aromatic compounds and highly unsaturated compounds toward compounds with lower O/C and higher H/C ratios. The observed molecular changes were most pronounced in organic compounds containing only CHO atoms. Our data thus, highlights classes of sedimentary organic compounds that may serve as microbial energy sources in methanic marine subsurface environments.

Geochemical data from pore water and solids from cores GS08-153-91GC12 and GS08-153-53-GC6, Arctic Mid-Ocean Ridge

Microbial communities and their associated metabolic activity in marine sediments have a profound impact on global biogeochemical cycles. Their composition and structure are attributed to geochemical and physical factors, but finding direct correlations has remained a challenge. Here we show a significant statistical relationship between variation in geochemical composition and prokaryotic community structure within deep-sea sediments. We obtained comprehensive geochemical data from two gravity cores near the hydrothermal vent field Loki's Castle at the Arctic Mid-Ocean Ridge, in the Norwegian-Greenland Sea. Geochemical properties in the rift valley sediments exhibited strong centimeter-scale stratigraphic variability. Microbial populations were profiled by pyrosequencing from 15 sediment horizons (59,364 16S rRNA gene tags), quantitatively assessed by qPCR, and phylogenetically analyzed. Although the same taxa were generally present in all samples, their relative abundances varied substantially among horizons and fluctuated between Bacteria- and Archaea-dominated communities. By independently summarizing covariance structures of the relative abundance data and geochemical data, using principal components analysis, we found a significant correlation between changes in geochemical composition and changes in community structure. Differences in organic carbon and mineralogy shaped the relative abundance of microbial taxa. We used correlations to build hypotheses about energy metabolisms, particularly of the Deep Sea Archaeal Group, specific Deltaproteobacteria, and sediment lineages of potentially anaerobic Marine Group I Archaea. We demonstrate that total prokaryotic community structure can be directly correlated to geochemistry within these sediments, thus enhancing our understanding of biogeochemical cycling and our ability to predict metabolisms of uncultured microbes in deep-sea sediments.

Effect of increased pCO2 on bacterial assemblage shifts in response to glucose addition in Fram Strait seawater mesocosms

Ocean acidification may stimulate primary production through increased availability of inorganic carbon in the photic zone, which may in turn change the biogenic flux of dissolved organic carbon (DOC) and the growth potential of heterotrophic bacteria. To investigate the effects of ocean acidification on marine bacterial assemblages, a two-by-three factorial mescosom experiment was conducted using surface sea water from the East Greenland Current in Fram Strait. Pyrosequencing of the V1-V2 region of bacterial 16S ribosomal RNA genes was used to investigate differences in the endpoint (Day 9) composition of bacterial assemblages in mineral nutrient-replete mesocosms amended with glucose (0 µm, 5.3 µm and 15.9 µm) under ambient (250 µatm) or acidified (400 µatm) partial pressures of CO2 (pCO2). All mesocosms showed low richness and diversity by Chao1 estimator and Shannon index, respectively, with general dominance by Gammaproteobacteria and Flavobacteria. Nonmetric multidimensional scaling analysis and two-way analysis of variance of the Jaccard dissimilarity matrix (97% similarity cut-off) demonstrated that the significant community shift between 0 µm and 15.9 µm glucose addition at 250 µatm pCO2 was eliminated at 400 µatm pCO2. These results suggest that the response potential of marine bacteria to DOC input may be altered under acidified conditions.

Small Changes in pH Have Direct Effects on Marine Bacterial Community Composition: A Microcosm Approach

As the atmospheric CO2 concentration rises, more CO2 will dissolve in the oceans, leading to a reduction in pH. Effects of ocean acidification on bacterial communities have mainly been studied in biologically complex systems, in which indirect effects, mediated through food web interactions, come into play. These approaches come close to nature but suffer from low replication and neglect seasonality. To comprehensively investigate direct pH effects, we conducted highly-replicated laboratory acidification experiments with the natural bacterial community from Helgoland Roads (North Sea). Seasonal variability was accounted for by repeating the experiment four times (spring, summer, autumn, winter). Three dilution approaches were used to select for different ecological strategies, i.e. fast-growing or low-nutrient adapted bacteria. The pH levels investigated were in situ seawater pH (8.15-8.22), pH 7.82 and pH 7.67, representing the present-day situation and two acidification scenarios projected for the North Sea for the year 2100. In all seasons, both automated ribosomal intergenic spacer analysis and 16S ribosomal amplicon pyrosequencing revealed pH-dependent community shifts for two of the dilution approaches. Bacteria susceptible to changes in pH were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. Their specific response to reduced pH was often context-dependent. Bacterial abundance was not influenced by pH. Our findings suggest that already moderate changes in pH have the potential to cause compositional shifts, depending on the community assembly and environmental factors. By identifying pH-susceptible groups, this study provides insights for more directed, in-depth community analyses in large-scale and long-term experiments.

Shallow water marine sediment bacterial community shifts along a natural CO2 gradient in the Mediterranean Sea Off vulcano, Italy

The effects of increasing atmospheric CO(2) on ocean ecosystems are a major environmental concern, as rapid shoaling of the carbonate saturation horizon is exposing vast areas of marine sediments to corrosive waters worldwide. Natural CO(2) gradients off Vulcano, Italy, have revealed profound ecosystem changes along rocky shore habitats as carbonate saturation levels decrease, but no investigations have yet been made of the sedimentary habitat. Here, we sampled the upper 2 cm of volcanic sand in three zones, ambient (median pCO(2) 419 µatm, minimum Omega (arag) 3.77), moderately CO(2)-enriched (median pCO(2) 592 µatm, minimum Omega (arag) 2.96), and highly CO(2)-enriched (median pCO(2) 1611 µatm, minimum Omega (arag) 0.35). We tested the hypothesis that increasing levels of seawater pCO(2) would cause significant shifts in sediment bacterial community composition, as shown recently in epilithic biofilms at the study site. In this study, 454 pyrosequencing of the V1 to V3 region of the 16S rRNA gene revealed a shift in community composition with increasing pCO(2). The relative abundances of most of the dominant genera were unaffected by the pCO(2) gradient, although there were significant differences for some 5 % of the genera present (viz. Georgenia, Lutibacter, Photobacterium, Acinetobacter, and Paenibacillus), and Shannon Diversity was greatest in sediments subject to long-term acidification (>100 years). Overall, this supports the view that globally increased ocean pCO(2) will be associated with changes in sediment bacterial community composition but that most of these organisms are resilient. However, further work is required to assess whether these results apply to other types of coastal sediments and whether the changes in relative abundance of bacterial taxa that we observed can significantly alter the biogeochemical functions of marine sediments.

Pore water measurements of sediment cores from the Helgoland mud area, North Sea

Iron reduction in subseafloor sulfate-depleted and methane-rich marine sediments is currently a subject of interest in subsurface geomicrobiology. While iron reduction and microorganisms involved have been well studied in marine surface sediments, little is known about microorganisms responsible for iron reduction in deep methanic sediments. Here, we used quantitative PCR (Q-PCR)-based 16S rRNA gene copy numbers and pyrosequencing-based relative abundances of bacteria and archaea to investigate covariance between distinct microbial populations and specific geochemical profiles in the top 5 m of sediment cores from the Helgoland mud area, North Sea. We found that gene copy numbers of bacteria and archaea were specifically higher around the peak of dissolved iron in the methanic zone (250-350 cm. The higher copy numbers at these depths were also reflected by the relative sequence abundances of members of the candidate division JS1, methanogenic and Methanohalobium/ANME-3 related archaea. The distribution of these populations was strongly correlated to the profile of pore-water Fe2+ while that of Desulfobacteraceae corresponded to the pore-water sulfate profile. Furthermore, specific JS1 populations also strongly co-varied with the distribution of Methanosaetaceae in the methanic zone. Our data suggest that the interplay among JS1 bacteria, methanogenic archaea and Methanohalobium/ANME-3-related archaea may be important for iron reduction and methane cycling in deep methanic sediments of the Helgoland mud area and perhaps in other methane-rich depositional environments. .